PROJECT SUMMARY/ABSTRACT Despite the success of antiretroviral therapy (ART) for HIV-1, new approaches that do not require continuous administration are clearly needed. This proposal will develop a technology platform to enable engineering of a novel approach, Therapeutic Interfering Particles (TIPs), which have the predicted capacity to be single- administration therapies that suppress HIV-1 set-point viremia. TIPs are engineered sub-genomic variants of HIV-1 that are trans-activated by HIV-1 gene expression and conditionally mobilize (i.e., `piggyback') with HIV- 1. TIPs thereby target the same cells that HIV-1 infects, stably transduce these cells, and remain latent until trans-activated by HIV-1?when HIV-1 infects or reverses latency in a TIP-transduced cell, TIPs act as molecular parasites, co-opting viral packaging resources to reduce HIV-1 burst size (i.e., virus emitted from a single cell). Based on our extensive theoretical evidence (Metzger et al., 2011; Rouzine et al. 2013; Rast et al. 2016), our central hypothesis is that TIPs, which (i) reduce HIV-1 `burst size' and (ii) conditionally mobilize to transduce new CD4+ T cells, would reduce set-point HIV-1 viremia, long term, by > 2-logs. The rationale for the mobilization premise derives from human clinical trials (Levine et al., 2006)?based on our theoretical predictions (Weinberger et al., 2003)?showing that lentiviral gene-therapies can be mobilized by HIV-1 in vivo. Computational modeling predicts that conditionally mobilizing TIPs would enable single-administration usage, thereby surmounting barriers faced by ART or potential vaccines (Metzger et al., 2011). TIPs are a fundamental departure from gene-therapy approaches and do not encode non-HIV-1 genetic elements or antiviral payloads, thereby circumventing problems of transgene mutation, reversion and deletion (ubiquitous problems in synthetic biology that lead to loss of efficacy) and limiting the generation of potentially dangerous viral recombinants. Our analyses show that TIPs are under evolutionary selection pressures similar to HIV-1 and co-evolve with HIV-1 to be potentially `resistance proof' (Rouzine and Weinberger, 2013). This proposal will test the hypothesis that TIPs constitute single-administration therapies by constructing an ultra-high- throughput sequencing assay to identify HIV-1 interference variants and testing TIP safety and efficacy in human patient isolates and cells as well as a non-human primate model of HIV infection. While the TIP approach carries inherent risks, successful completion of these studies will have broad significance by providing in vivo evidence to mitigate the safety risks and shift the HIV-1 treatment paradigm. Regardless of the success of TIPs, these studies will generate a general tool for fundamental virology by developing an assay for fundamental virology research to quantify changes in viral burst size, which will likely be useful for a broad array of viruses.